Cyclopeptide derivatives usable as selective inhibitors with respect to proteases with active serine

ABSTRACT

The invention relates to cyclopeptide derivative usable as protease inhibitors and complying with formula: ##STR1## in which R 1  is a halogen atom, e.g. bromine or a radical such as S +  R 2   4  X 1/v   v- , R 2  is a hydrogen atom, an alkyl radical, a halogen atom or other groups, R 3  is NH, O or S, Z comprises a peptide sequence Z 1  and a group such that CO has one end connected to the aromatic nucleus and CO-AA is a radical derived fropm an amino acid having a specificity with respect to the protease to be inhibited.

The present invention relates to novel cyclopeptide derivatives usableas protease inhibitors.

It more particularly applies to proteases with active cysteine or serinesuch as factors IXa, Xa, XIa, XIIa and VIIa of the coagulation of blood,thrombin, kallikrein of the plasma, activated protein C, activationfactors Clr, Cls, D and B in complement, C₃ convertase, trypsin,chymotrypsin, elastase, enterokinase, plasmin, activators of plasminogen(urokinase, tpA), acrosine, cathepsin G, chymases, tryptases andproteases dependent on ATP.

More specifically, it relates to novel cyclopeptide derivatives liableto act selectively as inhibitors with respect to a given protease.

It is known that active cysteine or serine proteases are involved innumerous physiological processes. Pathological states can occur whenthere is a non-equilibrium between a protease and its naturalmacromolecular inhibitors. In order to obviate this non-equilibrium,synthetic inhibitors could be used and would therefore have a greatinterest in therapeutics, e.g. in the following pathologies:

pulmonary emphysema, rheumatoid arthritis, cutaneous aging andinflammation, where the active protease is leucocytic elastase,cathepsin G also being involved;

tumor invasion and metastasis linked with the presence of proteases suchas activators of plasminogen or plasmin;

antithrombotic action and the prevention of cerebral and coronaryinfarctions, where the responsible protease is thrombin;

control of thrombolysis and fertility, relating to the activators ofplasminogen and plasmin;

control of parasites and viruses, because certain viruses produce activecysteine proteases.

For some years research has been carried out with the aim ofsynthesizing irreversible inhibitors of proteases. Among these are inparticular known synthetic inhibitors in the form of derivatives ofcoumarins, such as 3,4-dihydro-3,4-dibromo-6-bromomethyl coumarin, whichis a very effective, irreversible inactivator or inhibitor with respectto cysteine and serine proteases, but which unfortunately lacksselectivity. These coumarin derivatives are in particular described inthe book "Proteinase inhibitors" by A. J. Barret and G. Salvesen,Elsevier, 1986, pp. 119-121.

It would also be of great interest to synthesize other inhibitors ofproteases, which are specific with respect to a given protease and whichmake it possible to inactivate the latter without acting on analogousproteases.

The present invention specifically relates to a novel cyclopeptidederivative making it possible to achieve this result.

According to the invention, the cyclopeptide derivative corresponds tothe formula: ##STR2## in which: R¹ is chosen from among chlorine,fluorine, bromine and iodine atoms and the radicals OSO₂ R⁴, OP(O)R⁴ ₂,OC(O)R⁴ and S⁺ R⁴ ₂ X_(1/v) ^(v-) with R⁴ representing an alkyl,perfluoroalkyl or aryl radical, in which the R⁴ can be different and X⁻represents an anion of valency v;

R² is a hydrogen atom, an alkyl radical, a halogen atom, NO₂, COOR⁵,CF₃, CN or SO₂ R⁵ with R⁵ representing an alkyl or aryl radical;

R³ stands for an oxygen atom, a sulphur atom or --NH--;

Z comprises a peptide sequence Z₁ of amino acids or identical ordifferent analogs and a group chosen from among --(CH₂)_(n),--O(CH₂)_(n) -- or CO-- at its end connected to the aromatic nucleuswith n being an integer from 1 to 8; ##STR3## a radical derived from anamino acid or an amino acid analog of formula: ##STR4## in which R⁶represents --N-- or --CH-- and R⁷ represents a radical chosen from amongH, --CH₃, --CH₂ CH(CH₃)₂ ; ##STR5## CH₂ --CH₂ --COOH, --CH₂ --CH₂--CO--NH₂ --, (CH₂)₄ --NH₂, --CH₂ OH, --CH₂ --SH ##STR6## --CH₂ --C₆ H₄OH, --(CH₂)₃ NHC(═NH)--NH₂, --CH₂ --COOH, --CHOH--CH₃ ##STR7## or offormula: ##STR8## in which R⁸ represents N or CH and R⁹ represents theradical --(CH₂)₃ -- or --CH₂ --CHOH--CH₂.

The invention also relates to addition salts to pharmaceuticallyacceptable acids of the cyclopeptide derivatives. For example, it ispossible to use such salts when R⁷ comprises an amino group NH₂. Thepharmaceutically acceptable acids can e.g. be HCl, HBr, PO₄ H₃, R⁴ COOH,SO₄ H₂, R⁴ SO₃ H with R⁴ having the meaning given hereinbefore.

In this formula, the alkyl radicals used generally have 1 to 5 carbonatoms and can be straight or branched. The aryl radicals can have 6 to14 carbon atoms. Examples of such radicals are methyl, ethyl, phenyl andnaphthyl radicals.

In the cyclopeptide derivative according to the invention, the group CH₂R¹ makes it possible to inactivate the protease and the choice of thepeptide sequence Z₁, as well as the amino acid C(O)--AA makes itpossible to give the desired selectivity with respect to a givenprotease. Preferably, the peptide sequence Z₁ comprises 2 to 8 aminoacids or amino acid analogs, which can be the same or different.

The amino acids and amino acid analogs which can be used are e.g. thosecomplying with the formula ##STR9## in which R⁶ and R⁷ have the meaningsgiven hereinbefore.

Preferably, in the invention, R³ is in the ortho or meta position withrespect to Z and in the ortho or para position with respect to R¹ H₂ C.Thus, this arrangement makes it possible to obtain the best results withregards to the capacity of the group CH₂ R¹ to inactivate the proteaseand the capacity of the cyclopeptide chain to selectively adapt to theprotease to be inactivated.

The inactivation mechanism firstly corresponds to a cleaving of thecyclopeptide between R³ and the amino acid C(O)--AA, which permits afixing of the cyclopeptide to the active serine of the protease by thegroup C(O)--AA, whilst thus forming an acyl enzyme. This cyclopeptidecleaving gives R¹ an increased mobility by the formation of anintermediate derivative of the methylene quinonimine type, when R³represents NH, which is then transformed into methylene aniline withfixing to an amino acid residue of the active site of the enzyme by themethylene group, which thus ensures an inactivation of the enzyme.

Preferably R¹ is Cl, Br, OSO₂ R⁴ or preferably S⁺ R₄ ² X_(1/v) ^(v-). X⁻can be an anion derived from a pharmaceutically acceptable acid such asCl⁻, ClO₄ ⁻, Br⁻, BF₄ ⁻, PF₆ ⁻, R⁴ COO⁻, R⁴ SO₃ with R⁴ having themeaning given hereinbefore, e.g. CF₃ COO⁻. In S⁺ R² ₄ X_(1/v) ^(v-), thetwo R⁴ can be the same or different and are preferably alkyl or arylradicals.

In the cyclopeptide according to the invention, Z and AAC(O) are chosenas a function of the protease to be inactivated.

--AA--C(O)-- is derived from an amino acid, which corresponds to theprimary specificity of the targeted protease class.

In the case where it is wished to inactivate a protease of thechymotrypsin type, --AA--C(O)-- is preferably derived from phenylalanine, tyrosine, tryptophan or methionine and corresponds to theformula: ##STR10## in which R⁶ is CH and R⁷ is CH₂ --C₆ H₅, CH₂ --C₆ H₄OH, ##STR11## or (CH₂)₂ SCH₃.

In the case where the protease to be inhibited belongs to the trypsingroup, --AA--C(O)-- is preferably derived from lysine or arginine andcorresponds to the formula: ##STR12## in which R⁶ is CH and R⁷ is--(CH₂)₄ NH₂ or --CH₂)₃ NH--C(═NH)--NH₂.

When the protease to be inactivated belongs to the elastase type,--AA--C(O)-- is preferably derived from alanine or valine andcorresponds to the formula: ##STR13## in which R⁶ is CH and R⁷ is --CH₃or --CH(CH₃)₂.

According to the invention, the covalent chain Z is also chosen as afunction of the protease to be inactivated. This chain takes account ofthe affinity of the fixation subsites of the natural substrates of theprotease to be inactivated. Thus, the proteases have extensive activecentres and it is possible to increase the effectiveness and specificityof the inhibitor by choosing a covalent chain Z fulfilling thespecificity conditions of the fixation subsites.

In the case where the protease is of the trypsin, chymotrypsin orelastase type, it is possible to fulfil this specificity condition withZ complying with the formula: ##STR14## in which m=4,5 or 6 and R⁶ andR⁷ have the meanings given hereinbefore.

In the cyclopeptide according to the invention, the benzene nucleusforming part of the cyclopeptide can also have a substituent R², whichdoes not modify the inactivation mechanism of the protease by the groupCH₂. This substituent R² can be a hydrogen atom, a halogen atom ordifferent radicals, as shown hereinbefore.

The present invention also relates to pharmaceutical compositions havingthe property of inhibiting a given protease, which incorporate apharmaceutically acceptable quantity of a cyclopeptide derivativeaccording to the invention.

The pharmaceutical compositions can be in the form of solutions,suspensions, powders or solubilizable granules, syrups or elixirs, ear,nose or eye drops, tablets, gelatin capsules, aerosols, ointments,transdermal applications or suppositories, in dosed administration formscontaining non-toxic supports, adjuvants and excipients. The injectionscan be e.g. of the intravenous, intramuscular, subcutaneous,intradermal, intrasternal and intraarticular types, whilst infusion orinstillation methods (e.g. intratracheal) can be used.

The preparations for oral use can contain one or more sweetening,flavouring or preserving agents. Tablets contain the active molecule ofthe cyclopeptide derivative mixed with non-toxic and pharmaceuticallyacceptable excipients. Among the excipients, reference can e.g. be madeto inert diluents, such as calcium carbonate or sodium carbonate,calcium phosphate or sodium phosphate and lactose; agents permitting thegranulation and disintegration, e.g. corn starch, fixing agents, e.g.gelatin and starch; lubricating agents, e.g. talc or magnesium stearate,etc. The tablets may or may not be coated (e.g. with the aid of glyceroldistearate or monostearate) in order to delay their disintegration andabsorption.

The gelatin capsules can be in the form of a hard capsule containing theactive molecule mixed with an inert solid (kaolin, calcium carbonate),or a soft capsule in which the cyclopeptide derivative is mixed withwater or a fatty substance (e.g. liquid paraffin).

Aqueous suspensions containing cyclopeptide derivatives and appropriateexcipients, with optionally one or more preservatives (e.g.ethyl-p-hydroxybenzoate), colouring agents, sweetening agents andflavouring agents can be produced. Among the excipients, reference canbe made to suspending agents (e.g. methyl cellulose and acacia gum),dispersing or wetting agents, such as e.g. natural phosphatides (examplelecithin) or products for condensing ethylene oxide with various partialesters of fatty acids or aliphatic alcohols. Oily suspensions of theactive molecule can be prepared by using a vegetable oil (e.g. oliveoil) or a mineral oil (e.g. liquid paraffin), optionally in the presenceof sweetening and flavouring agents such as those referred tohereinbefore, as well as preservatives (in particular an antioxidant).

Syrups and elixirs could contain sweetening agents (e.g. sucrose andsorbitol), one or more preservatives and flavouring agents. Granules orpowders which can be suspended in water can be obtained by mixingcyclopeptide derivatives with a wetting or dispersing agent, one or morepreservatives and various excipients. Emulsions of cyclopeptidederivatives in water can be produced by using a mineral or vegetable oiland various emulsifiers, such as e.g. natural gums, natural phosphatidesand various esterified fatty acids.

The cyclopeptide derivatives can also be present in the form of aqueousor oily, sterile injectable suspensions using the suspending or wettingagents described hereinbefore. The solvents, diluents or excipients cane.g. be 1,3-butane diol, an isotonic sodium chloride solution, water,etc. The suppositories containing the active principle can be preparedwith conventional excipients in this field such as polyethylene glycolor coco butter. For local uses, ointments, creams, gels, suspensions,solutions, etc. containing the active principle can be prepared.

Doses of 0.1 to 40 mg/kg of body weight and day can be proposed, bearingin mind that the dose for a given patient can depend on a certain numberof factors, such as e.g. the efficacy of the cyclopeptide derivative inquestion, the age, the weight, the administration method, the diet,medicamentous interaction and the gravity of the illness.

These compositions can be used in the pathologies referred tohereinbefore. The cyclopeptide derivatives according to the inventioncan be prepared by conventional processes.

Thus, it is possible to prepare cyclopeptide derivatives in which R³represents NH from a derivative of formula (II): ##STR15## performingthe following successive steps:

a) Reaction of the compound of formula (II) with HZ¹ OC₂ H₅ to obtainthe compound of formula (III): ##STR16##

b) Reaction of the compound of formula (III) with hydrogen to obtain thecompound of formula (IV): ##STR17##

c) Reaction of the compound of formula (IV) with protected amino acidcorresponding to AA--C(O), which is e.g. in accordance with the formula:##STR18## in which Boc represents the tert butyl oxycarbonyl protectivegroup.

This reaction can be carried out using dicyclohexyl carbodiimide (DCC)and leads to the following compound of formula (V): ##STR19##

d) Reaction of the compound of formula V with hydrazine hydrate in asolvent such a methanol to obtain the compound of formula (VI):##STR20##

e) Reaction of the compound of formula (VI) with trifluoroacetic acid toobtain the compound of formula (VII): ##STR21##

f) Reaction of the compound of formula (VII) with nitrous acid or alkylnitrite to obtain the compound of formula (VIII): ##STR22##

g) Reaction of the compound of formula (VIII) with a tertiary amine suchas diisopropylethyl amine to obtain the cyclopeptide derivative offormula (IX): ##STR23##

From the compound of formula (IX) it is possible to obtain the compoundof formula (I) by reaction with appropriate reagents chosen as afunction of the nature of the group R¹ used.

In the case where R¹ is Br, the compound of formula (IX) is reacted withhydrobromic acid and acetic acid, which makes it possible to obtain thecompound of formula (X): ##STR24##

In the case where it is wished to obtain a cyclopeptide derivative offormula (I) with R¹ representing chlorine, the bromo derivative offormula (X) is reacted with sodium acetate in dimethyl formamide (DMF),which leads to the derivative of formula (XI): ##STR25## which is thentransformed into a derivative of formula (XII) by reaction with methanoland triethyl amine: ##STR26##

By then reacting the derivative of formula (XII) with thionyl chloride,the chloro derivative of formula (XIII) is obtained: ##STR27##

When it is wished to obtain the cyclopeptide derivative of formula (I),in which R¹ represents OSO₂ R⁴, the bromo derivative of formula (X) isreacted with the silver salt of sulphonic acid R⁴ SO₃ H, which makes itpossible to obtain the derivative of formula (XIV): ##STR28##

In the same way a silver salt of a phosphinic acid leads to derivativesin which R¹ =OP(O)R⁴ ₂ (formula I). The action of a dialkyl sulphide onthe bromo derivative gives the corresponding sulphonium salt (I; R¹ =S⁺R⁴ ₂).

However, preference is given to the preparation of cyclopeptidederivatives, in which R¹ represents S⁺ R⁴ ₂ X⁻ 1/v from thecorresponding derivatives of formula (IX) by reacting the latter withSR⁴ ₂ and the acid of formula XH_(v), in accordance with the followingreaction diagram: ##STR29##

For example, SR⁴ ₂ can represent thioanisole CH₃ SC₆ H₅ or dimethylsulphide CH₃ SCH₃.

The starting product of formula (II) can be obtained from thecorresponding methylated nitrobenzoic acid, following the esterificationof the acid function, by reacting with N-bromosuccinimide in order toform the derivative of formula (XV): ##STR30## which is then reactedwith a sodium phenate and then with potassium and then with SOCl₂ in thepresence of dimethyl formamide in order to form the compound of formula(II), in accordance with the following reaction diagram:

The invention will be better understood from studying the followingexamples which are given in an illustrative and non-limitative manner.

EXAMPLE 1 Preparation of the Cyclopeptide Derivative of Formula##STR32##

This formula corresponds to a cyclopeptide derivative of formula (I), inwhich R¹ represents bromine, R² a hydrogen atom, Z represents CO(Gly)₄,R⁴ represents NH and C(O)--AA is derived from lysine.

1) Preparation of the compound of formula (II) with R² =H; COCl in themeta position with respect to NO₂ and C₆ H₅ OCH₂ in the para positionwith respect to NO₂ ##STR33##

11.7 g (63.6 mmoles) of 3-nitro-6-methyl benzoic acid are esterifiedinto the corresponding methyl ester by an acetyl chloride excess inmethanol under reflux. The ester obtained is dissolved in 150 ml of CCl₄and treated with 12.5 g (70 mmoles) of N-bromosuccinimide (NBS) and 0.11g of benzoyl peroxide under reflux for 24 hours. The reaction mixture isfiltered on frit and the filtrate evaporated to dryness under reducedpressure. The crude product obtained of formula (BrCH₂) (NO₂) C₆ H₃--COOCH₃ is directly treated, without prior purification, by 12 g (127mmoles) of phenol and 2.50 g (6.2 mmoles) of aliquat (tricapryl methylammonium chloride) in 250 ml of CH₂ Cl₂, to which are added 5.08 g (127mmoles) of soda in pellet form dissolved in 250 ml of water. Thereaction mixture is vigorously stirred at ambient temperature for 5hours. The organic phase is then separated, washed with 400 ml of water,dried on MgSO₄, filtered, concentrated to approximately 150 ml and thenundergoes chromatography on a silica column (Merck silica gel 60),whilst eluting with methylene chloride (CH₂ Cl₂). A fraction ofapproximately 600 ml is collected, which is evaporated to dryness. Theresidue obtained is dissolved in 150 ml of CH₂ Cl₂. There is an additionof approximately 200 ml of methanol and refluxing takes place.Crystallization is observed in the boiling solution once all the CH₂ Cl₂has evaporated. The mixture is concentrated to 150 ml and left atambient temperature overnight. The white crystals are filtered on aBuchner filter, abundantly rinsed with methanol and dried in air. Thisgives 11.1 g of crystals corresponding to the derivative (C₆ H₅ OCH₂)(NO₂) C₆ H₃ --COOCH₃. The yield is 60% based on the starting acid (CH₃)(NO₂) C₆ H₃ --COOH. Melting point: 114° C. NMR ¹ H spectrum (CDCl₃/TMS): 8.93, d (J=2 Hz), 1H (ArH₁); 8.45, dd (J=2 Hz and 8 Hz); 1H(ArH₂); 8.07, d (J=8 Hz), 1H (ArH₃); 6.8 to 7.5, m, 5H (C₆ H₅ O); 5.58,s, 2H (ArCH₂ O); 4.00, s, 3H (COOCH₃).

    ______________________________________                                        Elementary analysis                                                                            C         H      N                                           ______________________________________                                        Calculated for C.sub.15 H.sub.13 NO.sub.5                                                      62.71     4.56   4.88                                        Found            62.92     4.31   4.98                                        ______________________________________                                    

3.59 g (12.5 mmoles) of the preceding ester are saponified by 25 ml of4N aqueous KOH (100 mmoles) in 100 ml of methanol. The mixture isstirred at ambient temperature for 4 hours. 150 ml of water are addedand the methanol is evaporated at 35° C. under reduced pressure. Theaqueous solution is extracted with 100 ml of CH₂ Cl₂ and then acidifiedby an excess of concentrated hydrochloric acid. The resultingprecipitate is dissolved in 400 ml of CH₂ Cl₂. The organic phase isseparated, washed with 200 ml of water, dried on MgSO₄, filtered andevaporated to dryness, leading to 2.79 g of acid of formula (C₆ H₅ OCH₂)(NO₂) C₆ H₃ --COOH obtained in the form of pale yellow crystals. Theyield is 82%. Melting point: 170° C. NMR ¹ H spectrum (acetone d₆):8.93, d (J=2 Hz), 1H (ArH₁); 8.55, dd (J=2 Hz and 8 Hz), 1H (ArH₂);8.15, d (J=8 Hz), 1H (ArH₃); 6.8 to 7.7, m, 5H (C₆ H₅ O--); 5.71, s, 2H(ArCH₂ O).

    ______________________________________                                        Elementary Analysis                                                                            C         H      N                                           ______________________________________                                        Calculated for C.sub.14 H.sub.11 NO.sub.5                                                      61.54     4.06   5.12                                        Found            61.98     3.84   5.12                                        ______________________________________                                    

2.79 g (10.2 mmoles) of the preceding acid are treated by 50 ml of SOCl₂and 10 drops of dimethyl formamide (DMF). The clear solution obtained isstirred for 2 hours at ambient temperature and then evaporated todryness under reduced pressure at 40° C. The residue obtainedcorresponds to acid chloride of formula (II): (C₆ H₅ OCH₂) (NO₂) C₆ H₃--CO--Cl and is directly used, without purification, in the followingstage (2).

2) Preparation of the compound of formula (XVII) ##STR34##

3.57 g (10 mmoles) of compound H--Gly₄ --OC₂ H₅ HBr and 50 ml of DMF arestirred and cooled to 0° C. 3 ml (21 mmoles) of triethyl amine areadded. The mixture is stirred for 10 minutes and then a solution of 10.2mmoles of acid chloride (II) with R² =H, COCl in the meta position withrespect to NO₂ and C₆ H₅ O--CH₂ in the para position with respect toNO₂, obtained in (1), in 40 ml of DMF is added dropwise in approximately30 minutes at 0° C. Following the addition, stirring is continued atambient temperature overnight. The mixture is evaporated to dryness at50° C. under reduced pressure. The residue obtained is triturated in 200ml of water and the resulting precipitate filtered on the Buchnerfilter, washed successively with 200 ml of H₂ O, 200 ml of 0.5N HCl, 100ml of H₂ O, 200 ml of 5% NaHCO₃ and 200 ml of H₂ O, dried in air andthen triturated in 50 ml of methanol, filtered on the Buchner filter,rinsed with methanol and then with ether, dried in air and this leads to3.83 g (yield 72%) of compound (XVII), whose melting point is 197° to200° C. and which is used as it is in the following stage (3). Ananalytical sample is prepared from 52 mg of the preceding sample,dissolved in 1 ml of DMF by crystallization following aqueous methanoladdition. 34 mg of crystals are obtained having a melting point of 199°to 203° C. Analysis by NMR ¹ H gives the following results: (DMSO-d.sub.6): 9.09, t (J=5.6 Hz), 1H (NH Gly); 8.42, d (J=2 Hz), 1H (ArH); 8.36,dd (J=2 Hz and 8.6 Hz), 1H (Ar Hz); 8.34, t (masked), 1H (NH Gly); 8.28,t (J 5.8 Hz), 1H (NH Gly); 8.23, t (J=5.8 Hz), 1H (NH Gly); 7.88, d(J=8.6 Hz), 1H (ArH₃); 7.3, m, 2H (C₆ H₅ O--); 7.0, m, 3H (C₆ H₅ --O--);5.39, s, 2H (C₆ H₅ O--CH₂ Ar); 4.06, q (J=7.1 Hz), 2H (O--CH₂ CH₃);3.96, d (J=5.5 Hz), 2H (CH₂ Gly); 3.81, d (J=5.5 Hz), 4H (2CH₂ Gly);3.75, d (J=5.6 Hz), 2H (CH₂ Gly); 1.17, t (J=7.1 Hz), 3H (OCH₂ CH₃).

    ______________________________________                                        Elementary Analysis  C       H       N                                        ______________________________________                                        Calculated for C.sub.24 H.sub.27 N.sub.5 O.sub.9 0.5 H.sub.2 O                                     53.52   5.24    13.00                                    Found                53.48   5.09    12.50                                    ______________________________________                                    

3) Preparation of the compound of formula (XIX) with lysine, whose αNH₂group is protected by a tert butyloxy carbonyl group Boc and whose εNH₂group is protected by a benzyloxy carbonyl group CBZ ##STR35##

Firstly, 1.06 g (2 mmoles) of the peptide of formula (XVII) aredissolved in 25 ml of DMF hot. This is followed by the addition of 75 mlof methanol and 150 mg (0.7 mmole) of PtO₂. The mixture is hydrogenatedfor 2 hours at ambient temperature, under 3 atmospheres using a Parrapparatus. The catalyst is filtered and the solvents evaporated underreduced pressure giving the compound of formula (XVIII): ##STR36## whosechromatographic analysis gives a R_(F) of 0.46 (support: silica, eluentCH₂ Cl₂ 85%--MeOH 15%). The presence of an aromatic amine function leadsto a fluorescent ultraviolet absorption at 366 nm of the chromatographicspot. This compound is not very stable in solution and is therefore usedimmediately after preparation, without further purification, for thesynthesis of compound (XIX).

Thus, the aromatic amine of formula (XVIII) previously obtained isdissolved in 1 ml of DMF, 25 ml of CH₂ Cl₂ are added and the solutioncooled to -5° C. This is followed by the addition of 0.912 g (2.4mmoles) of Nα-Boc-Nε-CBZ-L-lysine, then 0.495 g (2.4 mmoles) ofdicyclohexyl carbodiimide (DCC) in 25 ml of CH₂ Cl₂. The mixture isstirred at -5° C. for 2 hours and then at ambient temperature overnight.The dicyclohexyl urea precipitate formed is then filtered and thesolvents evaporated. The residue is dissolved in 400 ml of ethyl acetateand the organic phase is successively extracted with 200 ml of 5%NaHCO₃, 200 ml of H₂ O, 200 ml of 0.5N HCl, 200 ml of H₂ O, dried onMgSO₄, filtered and evaporated to dryness. The peptide obtained ispurified on a silica column (silica gel 60) using as the eluent amixture of methanol and CH₂ Cl₂ in proportions 10%:90%.

This gives the derivative of formula (XIX) with a 71% yield. Thechromatographic analysis on silica gives an R_(F) of 0.70 (eluent:methanol 15%--CH₂ Cl₂ 85%) or 0.29 (eluent: methanol 10%--CH₂ Cl₂ 90%).The melting point is 72° to 76° C. The rotary power is [α]₅₄₆ nm²⁵° C.=-9.1° (c 0.5; MeOH).

Analysis by NMR ¹ H gives the following results: (CD₃ OD): 8.07, s, 1H(ArH₁); 7.81, d (J=8.3 Hz), 1H (ArH₂); 7.71, d (J=8.3 Hz), 1H (ArH₃);7.51, m, 5H (ArH of CBZ); 7.44, m, 2H (C₆ H₅ O--); 7.13, m, 3H (C₆ H₅O--); 5.42, s, 2H (C₆ H₅ O--CH₂ --Ar); 5.23, s, 2H (CH₂ of CBZ); 4.35,m, 1H (CHαLys); 4.29, q (J=7.1 Hz), 2H (CH₂ of OEt); 4.24, s, 2H (CH₂Gly); 4.09, s, 2H (CH₂ Gly); 4.08, s, 2H (CH₂ Gly); 4.07, s, 2H (CH₂Gly); 3.31, m, 2H (CH₂ εLys); 1.91, m, 2H (CH₂ βLys); 1.70, m, 4H (CH₂ jand CH₂ δ of Lys); 1.63, s, 9H (3CH₃ of Boc); 1.40, t (J=7.1 Hz), 3H(CH₃ of OEt).

    ______________________________________                                        Elementary Analysis                                                                             C         H      N                                          ______________________________________                                        Calculated for C.sub.43 H.sub.55 N.sub.7 O.sub.12                                               59.92     6.43   11.38                                      Found             59.77     6.55   11.21                                      ______________________________________                                    

4) Preparation of the Derivative of Formula (XX) ##STR37##

3.2 g (3.7 mmoles) of ester of formula (XIX) in solution in 150 ml ofmethanol are stirred in the presence of 5.25 ml (108 mmoles) ofhydrazine hydrate H₂ NNH₂, H₂ O at ambient temperature for 12 hours. Thesolution is evaporated under reduced pressure at 40° C. The residue istaken up several times with methanol and evaporation to dryness takesplace until the hydrazine has been completely entrained. The solidresidue is triturated in ether and the supernatant removed. Afterdrying, 3.109 g of derivative (XX) is obtained in the form of a whitepowder. The yield is 98%. The melting point is 100° to 110° C.

Chromatographic analysis on a silica support gives a R_(F) of 0.46(eluent: methanol 20%--CH₂ Cl₂ 80%). The rotary power is [α]₅₄₆ ²⁵° C.=-8.4° (c 0.6; MeOH).

Analysis by NMR ¹ H gives the following results: (CD₃ OD/TMS): 7.88, d(J=1.8 Hz), 1H (ArH₁); 7.62, dd (J=1.8 Hz and 8.4 Hz), 1H (ArH₂); 7.53,d (J=8.4 Hz), 1H (ArH₃); 7.30, m, 5H (ArH of CBZ); 7.24, m, 2H (C₆ H₅O--); 6.94, m, 3H (C₆ H₅ O--)); 5.22, s, 2H (C₆ H₅ OCH₂ Ar); 5.04, s, 2H(CH₂ of CBZ); 4.15, m, 1H (CHα of Lys); 4.06, s, 2H (CH₂ of Gly); 3.88,s, 2H (CH₂ of Gly); 3.87, s, 2H (CH₂ of Gly); 3.84, s, 2H (CH₂ of Gly);3.12, m, 2H (CH₂ ε of Lys); 1.75, m, 2H (CH₂ β of Lys); 1.51, m, 4H (CH₂γ and CH₂ δ of Lys); 1.43, s, 9H (3CH₃ of Boc).

    ______________________________________                                        Elementary Analysis  C       H       N                                        ______________________________________                                        Calculated for C.sub.41 H.sub.53 N.sub.9 O.sub.11, 0.5 H.sub.2 O                                   57.46   6.35    14.71                                    Found                57.36   6.37    14.46                                    ______________________________________                                    

5) Preparation of the derivative of formula (XXI) ##STR38##

3.109 g (3.7 mmoles) of the compound of formula (XX) are dissolved in 80ml of glacial acetic acid and addition takes place of 20 ml of a 2Mhydrochloric acid solution in acetic acid. After stirring for 30minutes, 150 ml of ethyl ether are added. The precipitate is allowed tosettle and the supernatant removed. The residue is rinsed three timeswith ethyl ether, the supernatant being removed on each occasion aftersettling. The residual precipitate is dissolved in 100 ml of methanoland the solution is evaporated to dryness under reduced pressure at 40°C. This operation is repeated a second time. The residue is trituratedin ethyl ether until a homogeneous white powder is obtained. Thesupernatant ether is eliminated and the residual solid dried in vacuo.This gives 2.947 g of derivative of formula (XXI) in the form of whitepowder.

The yield is 98% and the melting point 84° to 88° C. (decomposition).Chromatographic analysis on a silica support gives an R_(F) of 0.07(eluent: MeOH 20%--CH₂ Cl₂ 80%) or 0.62 (eluent: ethylacetate/n-butanol/acetic acid/water in proportion 1:1:1:1).

The rotary power is [α]₅₄₆ ²⁵° C. =+23.0° (c 0.5; MeOH).

Analysis by NMR ¹ H gives the following results: (CD₃ OD/TMS): 7.93, d(J=2.1 Hz), 1H (ArH₁); 7.71, dd (J=2.1 Hz and 8.4 Hz) 1H (ArH₂); 7.56, d(J=8.4 Hz), 1H (ArH₃); 7.31, m, 5H (ArH of CBZ); 7.25, m, 2H (C₆ H₅O--); 6.94, m, 3H (C₆ H₅ O--); 5.24, s, 2H (C₆ H₅ O--CH₂ --Ar); 5.01, s,2H (CH₂ of CBZ); 4.07, s, 2H (CH₂ of Gly); 4.03, m, 1H (CHα of Lys);3.95, s, 2H (CH₂ of Gly); 3.91, s, 2H (CH₂ of Gly); 3.89, s, 2H (CH₂ ofGly); 3.14, m, 2H (CH₂ ε of Lys); 1.97, m, 2H (CH₂ β of Lys); 1.53, m,4H (CH₂ γ and CH₂ δ of Lys).

6) Preparation of the Cyclopeptide of Formula (XXII) ##STR39##

A solution of 0.820 g (1 mmole) of the compound of formula (XXI) isbrought into 20 ml of DMF at approximately -30° to -40° C. Acidificationthen takes place by the addition of 1.45 ml (8 mmoles) of a 5.5Mhydrochloric acid solution in THF, followed by the addition of 0.200 ml(1.5 mmole) of isoamyl nitrate. The solution is stirred at approximately-35° C. for 30 minutes and then diluted by adding 200 ml of cold DMF.The solution is alkalized to a pH of 8 to 9 by adding 2.1 cm³ (12mmoles) of diisopropyl ethyl amine, stirred at -35° C. for 10 minutesand then left to stand in a refrigerator (+4° C.) for 24 hours. Theseoperations are repeated several times in order to treat in all 3.13 g(3.82 mmoles) of the compound of formula (XXI).

The reaction mixtures are then combined and 200 ml of a 5% aqueous K₂CO₃ solution are added. Concentration takes place to approximately 150ml under a reduced pressure and at 40° C., in order to eliminate thediisopropyl ethyl amine, the water and part of the DMF. The mixture isthen filtered on frit to eliminate the insoluble mineral salts and thefiltrate is evaporated to dryness under reduced pressure at 40° C. Theresidue is an oil which crystallizes. By adding 50 ml of methanol, whitecrystals are obtained, which are filtered on a Buchner filter, rinsedwith methanol and dried (weight obtained 1.798 g). The filtrate isevaporated to dryness and the residue undergoes chromatography on asilica column (silica gel 60) using as the eluent a solution ofmethanol/CH₂ Cl₂ in a proportion of 15%:85%. The fractions containingthe product are combined and the solvents evaporated. The residue iscrystallized in methanol as previously. Thus, 0.076 g of crystals areobtained, i.e. in all 1.874 g of cyclopeptide of formula (XXII).

The yield is 68% and the melting point 270° to 280° C. (decomposition).Chromatographic analysis on a silica support gives an R_(F) of 0.55(eluent: MeOH 20%--CH₂ Cl₂ 80%) or 0.78 (eluent:EtOAc:1/nBuOH:1/AcOH:1/H₂ O:1). The corresponding spot is negative toninhydrin. The rotary power is [α]₅₄₆ ²⁵° C. =-40.6° (c 0.5; DMF).

Analysis by NMR ¹ H gives the following results: (DMSO d₆): 9.03, s, 1H(ArNHCO); 8.89, t (J=5.7 Hz), 1H (NH Gly); 8.75, t, (J=4.7 Hz), 1H (NHGly); 8.58, d (J=7.4 Hz), 1H (NH Lys); 8.20, dd (J=1.6 Hz and 8.4 Hz),1H (ArH₂); 8.16, t (J=6.0 Hz), 1H (NH Gly); 7.75, t (J=5.6 Hz), 1H (NHGly); 7.71, D (J=2.0 Hz), 1H (ArH₁); 7.50, d (J=8.5 Hz), 1H (ArH₃);7.34, m, 5H (ArH of CBZ); 7.29, m, 2H (C₆ H₅ O--); 6.94, m, 3H (C₆ H₅O); 5.21, s, 2H (C₆ H₅ OCH₂ --Ar); 5.00, s, 2H (CH₂ of CBZ); 4.20, m, 1H(CH α of Lys); 4.10 to 3.60, m, 8H (4CH₂ of Gly); 3.00, m, 2H (CH₂ ε ofLys); 1.93, m, 1H and 1.67, m, 1H (CH₂ β of Lys); 1.41, m, 4H (CH₂ γ andCH₂ δ of Lys).

    ______________________________________                                        Elementary Analysis                                                                             C         H      N                                          ______________________________________                                        Calculated for C.sub.36 H.sub.41 N.sub.7 O.sub.9                                                60.41     5.77   13.70                                      Found             60.40     5.70   13.43                                      ______________________________________                                    

7) Preparation of Cyclopeptide of Formula (XVI)

A mixture of 0.145 g (0.2 mmole) of cyclopeptide of formula (XXII) and20.4 ml of acetic acid is heated until a clear solution is obtained andis then cooled to ambient temperature. This is followed by the additionof 3.6 cm³ of a 33% HBr solution in acetic acid. The mixture is stirredat ambient temperature for 1 hour. 25 cm³ of ethyl ether are then added,which leads to the precipitation of the brominated cyclopeptide. Aftersettling the supernatant is removed. The precipitate is then stirred inether and then centrifuged, the supernatant then being removed. Thisoperation is repeated several times in order to eliminate all the acidsfrom the medium. The precipitate is then dried in vacuo. This gives0.131 g of cyclopeptide of formula (XVI) in the form of a white powder.The yield is 99% and the melting point 190° to 200° C. (decomposition).

Chromatographic analysis on a silica support gives a R_(F) of 0.43(eluent: EtOAc:1/nBuOH:1/AcOH:1/H₂ O:1). The spot is negative toninhydrin. The rotary power is [α]₅₄₆ ²⁵° C. =35.5° (c 0.2; MeOH).

Analysis by NMR ¹ H gives the following results: (CD₃ OD): 8.27, dd(J=2.2 Hz and 8.4 Hz), 1H (ArH₂); 7.93, d (J=2.2 Hz), 1H (ArH₁); 7.66, d(J=8.4 Hz), 1H (ArH₃); 5.03, d (J=10.1 Hz) and 4.98, d (J=8.1 Hz), 2H(BrCH₂ Ar); 4.59, dd (J=4.4 Hz and 9.7 Hz), 1H (CH α of Lys); 4.38, d(J=16.9 Hz) and 4.13, d (J=16.9 Hz), 2H (CH₂ of Gly); 4.30, s, 2H (CH₂of Gly); 4.24, d (J=16.9 Hz) and 4.13, d (J=16.9 Hz), 2H (CH₂ of Gly);4.15, d (J=15.8 Hz) and 4.01, d (J=15.8 Hz), 2H (CH₂ of Gly); 3.15, t(J=7.5 Hz), 2H (CH₂ ε of Lys); 2.25, m, 1H and 2.03, m, 1H (CH₂ β ofLys); 1.89, m, 2H (CH₂ δ of Lys); 1.74, m, 2H (CH₂ γ of Lys).

    ______________________________________                                        Elementary Analysis                                                                              C        H      N                                          ______________________________________                                        Calculated for C.sub.22 H.sub.31 N.sub.7 O.sub.6 Br.sub.2                                        40.69    4.81   15.10                                      Found              40.88    5.01   13.64                                      ______________________________________                                    

Mass spectrum (FAB>0): 568 (MH⁺ for ⁷⁹ Br); 570 (MH⁺ for ⁸¹ Br).

EXAMPLE 2

The properties of the cyclopeptide derivative of formula (XVI) arechecked and in particular its capacity to inactivate bovine trypsin orhuman urokinase using the methods of Kitz and Wilson (for trypsin andurokinase) and the method of Hart and O'Brien (for trypsin), 1973,published in Biochemistry, 12, pp. 2940-2945.

The apparent inactivation constant k_(i) /K_(i) is in this case:

53 M⁻¹ s⁻¹ for bovine trypsin and

4.2 M⁻¹ s⁻¹ for human urokinase.

However, this cyclopeptide derivative has no action on elastase.

EXAMPLE 3

Preparation of the cyclopeptide derivative of formula (XXIII): ##STR40##

This formula corresponds to a cyclopeptide derivative of formula (I) inwhich R¹ represents bromine, R² a hydrogen atom, Z represents CO(Gly)₄,R³ representing NH and CO --AA being derived from arginine.

An operating procedure similar to that of Example 1 is followed forpreparing this cyclopeptide derivative, except that in the third stagethe compound of formula (XVIII) is reacted with arginine, whose α NH₂group is protected by a butyloxycarbonyl group Boc. This gives thecyclopeptide of formula (XXIII), which has the followingcharacteristics:

R_(F) =0.52 (support: silica, eluent: EtOAc:1/nBuOH:1/AcOH:1/H₂ O:1).

Melting point: 205° to 215° C. (decomposition).

Rotary power: [α]₅₄₆ ²⁵° C. =-210° (c 0.2; MeOH).

Analysis by nuclear magnetic resonance: (CD₃ OD): 8.25, dd (J=2.0 Hz and8.4 Hz), 1H (ArH₂); 7.96, d (J=2.0 Hz), 1H (ArH₁); 7.66, d (J=8.4 Hz),1H (ArH₃); 5.01, s, 2H (BrCH₂ Ar); 4.61, dd (J=4.7 Hz and 9.2 Hz), 1H(CH α of Arg); 3.9 to 4.5, m, 8H (4CH₂ of Gly); 3.45, m, 2H (CH₂ δ ofArg); 2.28, m, 1H and 2.06, m, 1H (CH₂ β of Arg); 1.93, m, 2H (CH₂ γ ofArg).

Mass spectrum (FAB>0): 596 (MH⁺ for ⁷⁹ Br) and 598 (MH⁺ for ⁸¹ Br).

EXAMPLE 4

The properties of the cyclopeptide derivative of formula (XXIII) arechecked and in particular its capacity to inactivate bovine trypsin orhuman urokinase using the same methods as in Example 2 and working at25° C. with the 0.025M sodium phosphate buffer, 0.1M NaCl, 0.05% (v/v)Tween 80 and at pH 7.5 for human urokinase. The apparent inactivationconstant ki/Ki is 150 M⁻¹ s⁻¹ for bovine trypsin.

The apparent inactivation constant ki/Ki is 165 M⁻¹ s⁻¹ for humanurokinase. The inactivating or inhibiting properties of this derivativewith respect to human t-PA are checked at 25° C. in a 0.05M Tris buffer,0.038M NaCl, 0.01% (v/v) Tween 80 and pH 8.3. In this case ki/Ki is 0.3M⁻¹ s⁻¹.

However, this cyclopeptide derivative has no action on elastase.

EXAMPLE 5 Preparation of the Cyclopeptide Derivative of Formula (XXIV)##STR41##

This cyclopeptide derivative corresponds to a derivative of formula (I),in which R¹ represents bromine, R² a hydrogen atom, Z representsCO(Gly)₄, R³ represents NH and CO --AA is derived from phenyl alanine.

In order to prepare this cyclopeptide derivative, an operating proceduresimilar to that of Example 1 is followed, except that in the third stageuse is made of phenyl alanine, whose NH₂ group is protected by abutyloxycarbonyl group Boc. This gives the cyclopeptide derivative offormula (XXIV).

Its characteristics are as follows: R_(F) --0.50 (eluent: MeOH 20%--CH₂Cl₂ 80%); 0.69 (eluent: EtOAc: 1/nBuOH:1/AcOH: 1/H₂ O:1).

Analysis by nuclear magnetic resonance: (CD₃ OD): 8.15, dd (J =2.1 Hzand 8.5 Hz), 1H (ArH₂); 7.92, d (J=2.2 H), 1H (ArH₁); 7.66, d (J=8.5Hz), 1H (ArH₃); 7.47, m, 5H (ArH of Phe); 5.00, s, 2H (BrCH₂ Ar); 4.86,m, 1H (CH α Phe); 3.7 to 42, m, 8H (4CH₂ Gly); 3.59, m, 1H and 3.20, m,1H (CH₂ of Phe); (CD₃ COOD): 8.03, d (J=8.3 Hz), 1H (ArH₂); 7.79, s, 1H(ArH₁); 7.56, d (J=8.5 Hz), 1H (ArH₃); 7.36, m, 5H (ArH of Phe); 4.93,dd (J=5 Hz and 10 Hz), 1H (CH α of Phe), 4.86, s, 2H (BrCH₂ Ar); 4.43, d(J=16.4 Hz), 1H and 4.35, d (J=16.4 Hz), 1H (CH₂ of Gly); 4.34, d(J=16.5 Hz), 1H and 4.17, d (J=16.4 Hz), 1H (CH₂ of Gly); 4.27, d(J=16.6 Hz), 1H and 4.10, d (J=16.5 Hz), 1H (CH₂ of Gly); 4.09, d(J=16.0 Hz), 1H and 3.92, d (J=16.1 Hz), 1H (CH₂ of Gly); 3.47, dd (J=5Hz and 15 Hz), 1H and 3.17, dd (J=10 Hz and 15 Hz), 1H (CH₂ of Phe).

Melting point: 180° to 200° C. (decomposition)

Rotary power: [α]₅₄₆ ²⁵° C. =-46.7° (c 0.12; DMF).

    ______________________________________                                        Elementary Analysis  C       H       N                                        ______________________________________                                        Calculated for C.sub.25 H.sub.27 N.sub.6 O.sub.6 Br 3.5                                            46.16.2 O                                                                             5.26    12.92                                    Found                46.23   4.71    11.78                                    ______________________________________                                    

Mass spectrum (FAB>0): 586 M.sup.° for ⁷⁹ Br) and 588 (M.sup.° for ⁸¹Br).

EXAMPLE 6

The properties of the cyclopeptide derivative of formula (XXIV) arechecked and in particular its capacity to inactivate bovinechymotripysin using the same methods as in Example 2. The apparentinactivation constant ki/Ki is in this case 7 M⁻¹ s⁻¹.

EXAMPLE 7 Preparation of the Cyclopeptide Derivative of Formula (XXV)##STR42##

This derivative corresponds to a cyclopeptide derivative of formula (I),in which R¹ represents Cl, R² a hydrogen atom, Z represents CO--Gly₄, R³represents NH and CO--AA is derived from phenyl alanine.

The starting product is the cyclopeptide derivative of formula (XXIV)and it is successively treated by sodium acetate in dimethyl formamide(DMF) and then by triethyl amine in methanol and finally by thionylchloride.

To this end, 0.025 g (0.04 mmole) of cyclopeptide of formula (XXIV) arevigorously stirred at ambient temperature in the presence of 0.300 g (3mmoles) of potassium acetate and 3 ml of DMF. The mixture is thenevaporated to dryness under reduced pressure. This is followed by theaddition of 25 ml of a 20:80 methanol/methylene chloride mixture,followed by stirring for a few minutes, filtration and evaporation ofthe solvents. The residue undergoes chromatography on a silica column(silica gel 60), eluting with a mixture of methanol (20%) and CH₂ Cl₂(80%). The fractions are combined and the solvents evaporated. Theresidue is dissolved in 2 ml of methanol. Ether addition leads toprecipitation. The precipitate is centrifuged, rinsed with ether,centrifuged again and dried. This gives 0.018 g of acetate correspondingto formula (XXVI): ##STR43##

The compound of formula (XXVI) obtained in this way with a yield of 75%has the following characteristics: melting point 190° to 210° C.(decomposition); R_(F) (silica)=0.5 (eluent: MeOH 20%--CH₂ Cl₂ 80%);0.55 (eluent: EtOAc:1/nBuOH:1/AcOH:1/H₂ O:1).

Analysis by nuclear magnetic resonance: (CD₃ OD): 8.20, dd (J=2.1 Hz and8.4 Hz), 1H (ArH₂); 7.93, d (J=2.2 Hz), 1H (ArH₁); 7.64, d (J=8.4 Hz),1H (ArH₃); 7.47, m, 5H (ArH of Phe); 5.49, d (J=12.9 Hz) and 5.43, d(J=12.9 Hz), 2H (AcOCH₂ Ar); 4.85, dd (J=4.6 Hz and 10.1 Hz), 1H (CH αof Phe); 4.32, d (J=16.7 Hz), 1H and 4.09, d (J=16.9 Hz), 1H (CH₂ ofGly); 4.25, s, 2H (CH₂ of Gly); 4.24, d (J=17.1 Hz), 1H and 4.09, d(J=16.9 Hz) 1H (CH₂ of Gly); 4.01, d (J=15.6 Hz), 1H and 3.82, d (J=15.7Hz), 1H (CH₂ of Gly); 3.60, dd (J=4.5 Hz) and 14.0 Hz), 1H and 3.18, dd(J=10.3 Hz and 14.0 Hz), 1H (CH₂ of Phe); 2.25, s, 3H (CH₃ COO--).

Rotary power: [α]₅₄₆ ²⁵° C. =-36.9° (c 0.13; DMF).

Mass spectrum (FAB<0): 566 (M^(Q)); 600 (M--H--CH₃ COOH)⁻.

0.066 g (0.12 mmoles) of derivative of formula (XXVI) is dissolved in 18ml of methanol, followed by the addition of 2 ml of triethyl amine andstirring the solution at ambient temperature for 48 hours. The mixtureis then evaporated to dryness at 40° C. under reduced pressure and theresidue undergoes chromatography on a silica column (silica gel 60),whilst eluting with a mixture of MeOH: 20%/CH₂ Cl₂ : 80%. The adequatefractions are combined and the solution concentrated to approximately 2ml. The addition of 15 ml of ethyl ether causes a precipitation. Theprecipitate is centrifuged, triturated in ether and centrifuged again,followed by drying in vacuo. This gives 0.043 g of derivative of formula(XXVII) with a yield of 70%: ##STR44##

The derivative of formula (XXVII) has the following characteristics:

Melting point: 200° to 220° C. (decomposition); R_(F) (silica): 0.50(eluent: MeOH 20%--CH₂ Cl₂ 80%);

rotary power: [α]₅₄₆ ²⁵° C. =-80.0° (c 0.2; DMF).

Analysis by nuclear magnetic resonance: (CD₃ OD): 8.23, dd (J=2.2 Hz and8.3 Hz), 1H (ArH₂); 8.00, d (J=2.2 Hz), 1H (ArH₁) 7.63, d (J=8.3 H_(X)),1H (ArH₃); 7.49, m, 5H (ArH of Phe); 4.87, s, 2H (OCH₂ Ar); 4.85, ddpartly masked (J=4.4 Hz and estimated 10.6 Hz), 1H (CH α of Phe); 4.38,d (J=16.8 Hz), 1H and 4.11, d (J=16.8 Hz), 1H (CH₂ of Gly); 4.30, d(J=16.3 Hz), 1H and 4.23, d (J=16,3 Hz), 1H (CH₂ of Gly); 4.28, d(J=17.1 Hz), 1H and 4.00, d (J=17.1 Hz), 1H (CH₂ of Gly); 4.03, d(J=15.4 Hz), 1H and 3.78, d (J=15.4 Hz), 1H (CH₂ Gly); 3.65, dd (J=4.3Hz and 14.2 Hz), 1H and 3.17, dd (J=10.6 Hz and 14.2 Hz), 1H (CH₂ ofPhe).

Finally, 0.0157 g (0.03 mmole) of the derivative of formula (XXVII) isdissolved in 0.5 ml of DMF. The solution is stirred at 0° C. and to itis added 0.25 ml of a freshly prepared solution maintained at 0° C. of0.17 ml of SOCl₂ in 5 ml of DMF. The mixture is stirred for 1 minute at0° C. and then 3 minutes at ambient temperature and is then immediatelyevaporated to dryness under reduced pressure. These operations arerepeated several times to treat in all 0.0443 g (0.08 mmole) of thederivative of formula (XXVII). The residues obtained after evaporationof the solvents are combined.

Purification takes place by silica column chromatography, whilst elutingwith a mixture of MeOH 15%--CH₂ Cl₂ 85%. The adequate fractions arecombined and the solution evaporated to dryness. The residue isdissolved in 2 ml of methanol and precipitated by ethyl ether addition.The precipitate is centifuged, triturated in ether, centrifuged againand dried in vacuo. This gives 0.0268 g of chloride of formula (XXV)with a yield of 58%. The derivative of formula (XXV) has the followingcharacteristics: melting point: 170° to 230° C. (decomposition in thesolid state);

R_(F) (silica): 0.67 (eluent: MeOH 20%--CH₂ Cl₂ 80%); 0.74 (eluent:EtOAc:1/nBuOH:1/AcOH:1/H₂ O:1).

Rotary power: [α]₅₄₆ ²⁵° C. =-31.8° (c 0.8; MeOH).

Analysis by nuclear magnetic resonance: (CD₃ OD): 8.17, dd (J=2.1 Hz and8.4 Hz), 1H (ArH₂); 7.94, d (J=2.1 Hz), 1H (ArH₁); 7.68, d (J=8.4 Hz),1H (ArH₃); 7.48, m, 5H (ArH of Phe); 4.86 dd (J=4.7N_(Z) and 10.0 Hz),1H (CH α of Phe); 4.83, s, 2H (ClCH₂ Ar); 4.32, d (J=17.0 Hz), 1H and4.12, d (J=17.0 Hz), 1H (CH₂ of Gly); 4.29, s, 2H (CH₂ of Gly); 4.23, d(J=16.9 Hz), 1H and 4.09, d (J=16.8 Hz), 1H (CH₂ of Gly); 4.01, d(J=15.8 Hz), 1H and 3.85, d (J=15.8 Hz), 1H (CH₂ of Gly); 3.59, dd(J=4.7 Hz and 14.0 Hz), 1H and 3.19, dd (J=10.1 Hz and 14.0 Hz), 1H (CH₂of Phe).

    ______________________________________                                        Elementary Analysis     C      H                                              ______________________________________                                        Calculated for C.sub.25 H.sub.27 N.sub.6 O.sub.6 Cl, 1.5                                              52.68.2 O                                                                            5.31                                           Found                   52.43  4.69                                           ______________________________________                                    

Mass spectrum (DCI): 542 (M° for ³⁵ Cl); 544 (M° for ³⁷ Cl).

EXAMPLE 8

The properties of the cyclopeptide derivative of formula (XXV) arechecked and in particular its capacity to inactivate bovine chymotrypsinby using the same methods as in Example 2. The apparent inactivationconstant k_(i) /K_(i) is in this case 2.8M⁻¹ s⁻¹.

The results of Examples 2,4,6 and 8 are summarized in the attached Table1.

EXAMPLE 9 Preparation of the Cyclopeptide of Formula (XXVIII) ##STR45##

A solution of 0.100 g (0.14 mmole) of cyclopeptide of formula (XXII) and1.412 cm³ (14 mmoles) of thioanisole in 5.6 cm³ of trifluoroacetic acid(TFA) is stirred at ambient temperature for 24 h. This is followed bythe addition of 50 cm³ of ethyl ether, which leads to the formation of awhite precipitate. The precipitate is decanted and the supernatantremoved. The precipitate is rinsed several times with ether, collectedby centrifuging, dried in vacuo and purified by silica columnchromatography (silica gel 60), whilst eluting with a solutionEtOAc/nBuOH/AcOH/H₂ O 1:1:1:1. The adequate fractions are combined andthe solution evaporated to dryness under reduced pressure. The residueobtained is dissolved in 2 cm³ of trifluoroacetic acid and thenprecipitated with ether. The precipitate is rinsed several times withether, collected by centrifuging and dried. It is dissolved in 2 cm³ ofmethanol, reprecipitated with ether, rinsed several times with ether,collected by centrifuging and dried. It is then dissolved in 10 cm³ ofwater. The solution is filtered and then lyophilized. This gives 0.056 gof cyclopeptide of formula (XXVIII) in the form of a white powder. Theyield is 48% and the melting point 135° C. (with decomposition).Chromatographic analysis on a silica support gives a Rf of 0.12 (eluentEtOAc/nBuOH/AcOH/H₂ O 1:1:1:1). The rotary power is (α)²⁵° C. (546nm)=-25.8° (c 0.2; H₂ O). Analysis by NMR ¹ H gives the followingresults: (D₂ O): 7.8-7.3, m, 3H (ArH¹ H² H³); 7.60, m, 5H (C₆ H₅ S⁺);5.13, m, 2H (ArCH₂ S⁺); 4.26, m, 1H (CHαLys); 3.94, m, 8H (4 CH₂ Gly);3.29, s, 3H (CH₃ S⁺); 2.96, m, 2H (CH₂ εLys); 1.9-1.4, m, 6H (CH₂βγδLys).

EXAMPLE 10 Preparation of the Cyclopeptide of Formula (XXIX) ##STR46##

This formula corresponds to a cyclopeptide derivative of formula (I), inwhich R¹ represents C₆ H₅ S⁺ (CH₃)--, CF₃ COO⁻ ; R² a hydrogen atom, Zrepresents CO(Gly)₄, R³ represents NH and C(O)--AA is derived fromarginine. An operating procedure similar to that of Example 1 (stages 1to 6) and Example 9 is followed, except that in the third stage ofExample 1, the compound of formula (XVIII) is reacted with arginine,whose αNH₂ group is protected by a butyloxycarbonyl group Boc. Thisgives the cyclopeptide of formula (XXIX), which has the followingcharacteristics: Rf=0.14 (silica support; eluent EtOAc/nBuOH/AcOH/H₂ O1:1:1:1). The melting point is 125° to 130° C. (with decomposition). Therotary power is (α)²⁵° C. (546 nm)=-4° (c 0.1; H₂ O). Analysis bynuclear magnetic resonance: ¹ H (D₂ O): 7.8-7.3, m, 3H (ArH¹ H² H³);7.61, m, 5H (C₆ H₅ S⁺); 5.10, m, 2H (ArCH₂ S⁺); 4.28, m, 1H (CHαArg);3.95, m, 8H (4 CH₂ Gly); 3.30, s, 3H (CH₃ S⁺); 3.20, m, 2H (CH₂ δArg);2.0-1.6, m, 4H (CH₂ βγArg).

EXAMPLE 11 Preparation of the Cyclopeptide of Formula (XXIX') ##STR47##

This compound is a position isomer of compound (XXIX). Its formulacorresponds to a cyclopeptide derivative of formula (I), in which R¹represents C₆ H₅ S⁺ (CH₃)--, CF₃ COO⁻, R² a hydrogen atom, Z representsCO(Gly)₄ in the ortho position (instead of the meta position) withrespect to R³, which represents NH and C(O)--AA is derived fromarginine. An operating procedure similar to that of Example 10 isfollowed except that the arginine, whose αNH₂ group is protected by aBoc group, is coupled to the compound of formula (XVIII') obtained inthe same way as (XVIII) from compound (II'), which is identical to (II)and which is itself obtained from 2-nitro-5-methyl-benzoic acid.##STR48##

This gives the cyclopeptide of formula (XXIX'), which has the followingcharacteristics: Rf=0.12 (silica support; eluent EtOAc/nBuOH/AcOH/H₂ O1:1:1:1). Melting point 140° to 150° C. (with decomposition). Rotarypower (α)²⁵° C. (546 nm)=+7.37° (c 0.1; H₂ O). Analysis by nuclearmagnetic resonance: ¹ H (D₂ O): 7.7-7.1, m, 8H (ArH¹ H² H³ and C₆ H₅S⁺); 4.60, m, 2H (ArCH₂ S⁺); 4.30, m, 1H (CHαArg); 3.90, m, 8H (4 CH₂Gly); 3.30, s, and 3.19, s, 3H (CH₃ S⁺); 3.20, m, 2H (CH₂ δArg);2.1-1.5, m, 4H (CH₂ βγArg).

EXAMPLE 12 Preparation of the Cyclopeptide of Formula (XXX) ##STR49##

This formula corresponds to a cyclopeptide of formula (I), in which R¹represents (CH₃)₂ s⁺, CF₃ COO⁻, R² a hydrogen atom, Z representsCO(Gly)₄, R³ represents NH and C(O)--AA is derived from arginine. Anoperating procedure similar to that of Example 10 is followed, exceptthat in the final stage the thioanisole is replaced by dimethylsulphide. This gives the cyclopeptide of formula (XXX), which has thefollowing characteristics: Rf=0.09 (silica support; eluentEtOAc/nBuOH/AcOH/H₂ O 1:1:1:1:1). Melting point 130° to 150° C. (withdecomposition). Rotary power (α)²⁵° C. (546 nm)=-25.7° (c 0.25; H₂ O).Analysis by nuclear magnetic resonance ¹ H (D₂ O); 7.75, dd (J=2.1 Hzand 8.3 Hz), 1H (ArH²); 7.63, d (J=2.1 Hz), 1H (ArH¹); 7.54, d (J=8.3Hz), 1H (ArH³); 4.68, s, 2H (ArCH₂ S⁺); 4.27, m, 1H (CH αArg); 4.2-3.8,m, 8H (4 CH₂ Gly); 3.19, t (J=6.6 Hz), 2H (CH₂ δArg); 2.80, s, 6H ([CH₃]₂ S⁺); 2.0-1.5, m, 4H (CH₂ βγArg).

EXAMPLE 13 Preparation of the Cyclopeptide of Formula (XXXI): ##STR50##

This formula corresponds to a cyclopeptide derivative of formula (I), inwhich R¹ represents bromine, R² represents H, Z represents CO-Gly₂-Pro-Gly, R³ represents NH and CO-AA is derived from arginine.

The operating procedure is identical to that of Example 3, except thatin the second stage the compound of formula (II) is reacted with thecompound H-Gly₂ -Pro-Gly OC₂ H₅ to obtain the compound of formula:##STR51## At the end of the final stage, the cyclopeptide of formula(XXXI) is obtained.

The rotary power is [α]₅₄₆ nm²⁵° C. =-31.1 (c 0.54; H₂ O).

Analysis by NMR ¹ H gives the following results: (D₂ O): 7.54, d, (J=1.6Hz), 1H (ArH₁); 7.51, d (J=8 Hz), 1H (ArH₃); 7.48, dd (J=8 Hz and 1.6Hz), 1H (ArH₂); 4.65, s, 2H (CH₂ Br).

EXAMPLE 14

The inactivating or inhibiting properties of the functionalizedcyclopeptides of formulas XXVIII, XXIX, XXIX', XXX and XXXI of Examples9 to 13, summarized in Table 2 were tested with respect to a certainnumber of enzymes: bovine trypsin, human urokinase, human t-PA, humanplasmin and porcine pancreatic elastase. The kinetic measurements werecarried out at 25° C. in the following buffers: 0.1M Tris, 0.01M CaCl₂,pH 7.2 (bovine trypsin); 0.025M sodium phosphate, 0.1M NaCl, 0.05% (v/v)Tween 80, pH 7.5 (human urokinase); 0.05M Tris, 0.038M NaCl, 0.01% (v/v)Tween 80, pH 8.3 (human t-PA), 0.1M Tris (porcine pancreatic elastase),0.1M sodium phosphate, 25% (v/v) glycerol (human plasmin). By usingeither the method of Hart and O'Brien, designated a (Biochemistry, 1973,Vol. 12, pp. 2940-2945) and/or that of Kitz and Wilson, designated by(J. Biol. Chem., 1962, Vol. 237, 32, pp. 3245-3249), the first orderconstant k_(i) was determined which characterizes the infinite substrateconcentration inactivation and the dissociation constant K_(i) of theenzyme-inhibitor complex. Their ratio k_(i) /K_(i) is an apparent secondorder constant characterizing the effectiveness of the inactivator. Theresults obtained are given in Table 2.

On the basis of the results of Tables 1 and 2, it can be seen that thecyclopeptide derivatives of Examples 9 to 13 are better inhibitors thanthe corresponding bromides of formulas XVI and XXIII of Examples 1 and3.

Moreover, the cyclopeptide derivatives of Examples 9 to 12 are morestable in aqueous solution than the bromo derivatives of Examples 1 and3.

Thus, the cyclopeptide derivatives according to the invention areselective inhibitors of certain proteases and can be used as the activesubstance in pharmaceutical compositions, which can be used intherapeutical applications in the pathologies referred to hereinbefore.

                  TABLE 1                                                         ______________________________________                                        Kinetic parameters characterizing the inactivation of serine                  proteases by functionalized cyclopeptides (25° C.).                                                k.sub.i /K.sub.i                                  Cyclopeptide     Enzyme     (M.sup.-1 s.sup.-1)                               ______________________________________                                        Compound (XXV) of                                                                              Bovine     2.8                                               Example 7        chymotrypsin                                                 Compound (XXIV) of                                                                             Bovine     7                                                 Example 5        chymotrypsin                                                 Compound (XVI) of                                                                              Bovine     53                                                Example 1        trypsin                                                                       Human      4.2                                                                urokinase                                                    Compound (XXIII) of                                                                            Bovine     150                                               Example 3        trypsin                                                                       Human      165                                                                urokinase                                                                     Human t-PA 0.3                                               ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Kinetic parameters characterizing the inactivation of serine                  proteases by functionalized cyclopeptides (25° C.).                                            k.sub.i /K.sub.i                                      Cyclopeptide Enzyme 1)  (M.sup.-1 s.sup.-1)                                                                     Method 2)                                   ______________________________________                                        Derivative (XXVIII)                                                                        Trypsin    490       a                                           of Example 9 Urokinase  145       b                                                        Plasmin    53        b                                                        Elastase   0                                                     Derivative (XXIX)                                                                          Trypsin    824       a                                           of Example 10                                                                              Urokinase  1967      a and b                                                  t-PA       1.4       b                                                        Elastase   0         b                                           Derivative (XXIX')                                                                         Trypsin    16.7      a                                           of Example 11                                                                              Urokinase  14        a                                                        Elastase   0         b                                           Derivative (XXX)                                                                           Trypsin    7         b                                           of Example 12                                                                              Urokinase  108       b                                                        Elastase   0         b                                                        t-PA       0         b                                           Derivative (XXXI)                                                                          Trypsin    2016      a                                           of Example 13                                                                 ______________________________________                                         1) Enzymes:                                                                   Trypsin: bovine trypsin                                                       Urokinase: human urokinase                                                    tPA: tissue activator of human plasminogen                                    Plasmin: human plasmin                                                        Elastase: porcine pancreatic elastase                                         2) Methods                                                                    a: Hart and O'Brien method.                                                   b: Kitz and Wilson method.                                               

We claim:
 1. Cyclopeptide derivative usable as a protease inhibitor,characterized in that it complies with the formula: ##STR52## in which:R¹ is chosen from among chlorine, fluorine, bromine and iodine atoms andthe radicals OSO₂ R⁴, OP(O)R⁴ ₂, OC(O)R⁴ and S⁺ R⁴ ₂ X_(1/v) ^(v-) withR⁴ representing an alkyl, perfluoroalkyl or aryl radical, in which theR⁴ can be different and X⁻ represents an anion of valency v;R² is ahydrogen atom, an alkyl radical, a halogen atom, NO₂, COOR⁵, CF₃ CN orSO₂ R⁵ with R⁵ representing an alkyl or aryl radical; R³ stands for anoxygen atom, a sulphur atom or --NH--; Z comprises a peptide sequence Z₁of amino acids or identical or different analogs and a group chosen fromamong --(CH₂)_(n), --O(CH₂)_(n) -- or CO-- at its end connected to thearomatic nucleus with n being an integer from 1 to 8; ##STR53## aradical derived from an amino acid or an amino acid analog of formula:##STR54## in which R⁶ represents --N-- or --CH-- and R⁷ represents aradical chosen from among H, --CH₃, --CH₂ CH(CH₃)₂ ; ##STR55## CH₂ --CH₂--COOH, --CH₂ --CH₂ --CO--NH₂ --(CH₂)₄ --NH₂, --CH₂ OH, --CH₂ --SH##STR56## --CH₂ --C₆ H₄ OH, --(CH₂)₃ NHC(=NH)--NH₂, --CH₂ --COOH,--CHOH--CH₃ ##STR57## or of formula: ##STR58## in which R⁸ represents Nor CH and R⁹ represents the radical --(CH₂)₃ -- or --CH₂ --CHOH--CH₂, aswell as its addition salts to a pharmaceutically acceptable acid. 2.Cyclopeptide derivative according to claim 1, characterized in that thepeptide sequence Z₁ comprises 2 to 8 amino acids or amino acid analogs,which can be the same or different.
 3. Cyclopeptide derivative accordingto one of the claims 1 or 2, characterized in that R³ is in the ortho ormeta position with respect to Z and in the ortho or para position withrespect to R¹ H₂ C.
 4. Cyclopeptide derivative according to claim 1usable as a trypsin inhibitor, characterized in that --AA--C--(O)--complies with the formula: ##STR59## in which R⁶ is CH and R⁷ is--(CH₂)₄ NH₂ or --(CH₂)₃ NH--C(═NH)--NH₂.
 5. Cyclopeptide derivativeaccording to claim 4, characterized in that R¹ is Cl, Br or OSO₂ R⁴. 6.Cyclopeptide derivative according to claim 1, characterized in that R¹represents S⁺ R⁴ ₂ X_(1/v) ^(v-).
 7. Cyclopeptide derivative accordingto claim 6, characterized in that R¹ represents ##STR60## 8.Cyclopeptide derivative according claim 4, characterized in that R² is ahydrogen atom.
 9. Cyclopeptide derivative according claim 2,characterized in that Z complies with the formula: ##STR61## with m=4, 5or 6 and R⁶ and R⁷ having the meanings given in claim
 1. 10.Pharmaceutical composition having the property of inhibiting a givenprotease, characterized in that it comprises a pharmaceuticalyacceptable quantity of a cyclopeptide derivative according to claim 1.11. Composition according to claim 10, characterized in that theprotease belongs to the group comprising trypsin, chymotrypsin, elastaseor active cysteine proteases.
 12. Cyclopeptide derivative according toclaim 2 wherein the peptide sequence Z₁ comprises 4 aminoacidsconsisting of glycine.